Microcapsules are one of the most promising microscale drug carriers due to their facile fabrication, excellent deformability, and high efficacy in drug storage and delivery. Understanding the effects of their physicochemical properties (size, shape, rigidity, charge, surface chemistry, etc.) on both in vitro and in vivo performance is not only highly significant and interesting but also very challenging. Stiffness, an important design parameter, has been extensively explored in recent years, but how the rigidity of particles influences cellular internalization and uptake mechanisms remains controversial. Here, one-layered lysozyme-based microcapsules with well-controlled stiffness (modulus ranging from 3.49 ± 0.18 MPa to 26.14 ± 1.09 MPa) were prepared and used to investigate the effect of stiffness on the uptake process in dendritic cells and the underlying mechanism. The cellular uptake process and endocytic mechanism were investigated with laser scanning confocal microscopy, mechanism inhibitors, and pathway-specific antibody staining. Our data demonstrated that the stiffness of protein-based microcapsules could be a strong regulator of intracellular uptake and endocytic kinetics but had no obvious effect on the endocytic mechanism. We believe our results will provide a basic understanding of the intracellular uptake process of microcapsules and the endocytic mechanism and inspire strategies for the further design of potential drug delivery microcarriers.